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The compound (ButO)3W identical to N serves to exchange the nitrogen atoms between nitriles (MeC identical to N and PhC identical to N), itself and (ButO)3Mo identical to N in solution at room temperature.

EB1 is a microtubule associated protein which interacts with the APC tumour suppressor protein and components of the cytoplasmic dynein/dynactin complex. EB1 is also a specific marker of growing microtubule tips. Here we demonstrate that EB1 protein levels are increased during axon but not dendrite formation in differentiated N2A neuroblastoma cells, and that EB1 localises to microtubule tips throughout extending neurites in these cells. In N2A axons, analysis of the ratio of EB1/β-tubulin fluorescence demonstrated that the distal tip region contained the highest proportion of polymerising microtubules. Time-lapse confocal imaging of an EB1-GFP fusion protein in transfected N2A cells directly revealed the dynamics of microtubule extension in neurites, and demonstrated the existence of unusual, discrete knots of microtubule polymerisation at the periphery of non-process bearing cells which may represent an early event in neurite outgrowth. We conclude that EB1 localisation can be used to identify and analyse sites of microtubule polymerisation at a high resolution during neurite development, a process to which it may contribute.

Summary Spindle positioning in animal cells is thought to rely upon the interaction of astral microtubules with the cell cortex. Information on the dynamics of astral microtubules during this process is scarce, in part because of the difficulty in visualising these microtubules by light microscopy. EB1 is a protein which specifically localises to growing microtubule distal tips. Immunostaining for EB1 therefore represents a powerful method for visualising the distribution of growing microtubule tips within cells. In this study we used EB1 immunostaining in mitotic NRK-52E cells to quantitatively analyse the length and number of growing astral microtubules during metaphase and anaphase. We observed a dramatic increase in growing astral microtubule length and number during anaphase. Furthermore, drug treatments which specifically destroyed astral microtubules resulted in an increase in misaligned anaphase but not metaphase spindles. We suggest that an anaphase-specific increase in growing astral microtubule length and number facilitates the maintenance of a correctly aligned spindle in mitotic NRK-52E cells.

Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.

The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse re-distribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells.

A linear supramoleculecular array of anions and cations is present in the solvent-free potassium salt of the homoleptic erbium pyrazolate complex [K{Er(η

The role of phosphorylation in the dissociation of structural components of the herpes simplex virus type 1 (HSV-1) tegument was investigated, using an in vitro assay. Addition of physiological concentrations of ATP and magnesium to wild-type virions in the presence of detergent promoted the release of VP13/14 and VP22. VP1/2 and the UL13 protein kinase were not significantly solubilized. However, using a virus with an inactivated UL13 protein, we found that the release of VP22 was severely impaired. Addition of casein kinase II (CKII) to UL13 mutant virions promoted VP22 release. Heat inactivation of virions or addition of phosphatase inhibited the release of both proteins. Incorporation of radiolabeled ATP into the assay demonstrated the phosphorylation of VP1/2, VP13/14, VP16, and VP22. Incubation of detergent-purified, heat-inactivated capsid-tegument with recombinant kinases showed VP1/2 phosphorylation by CKII, VP13/14 phosphorylation by CKII, protein kinase A (PKA), and PKC, VP16 phosphorylation by PKA, and VP22 phosphorylation by CKII and PKC. Proteolytic mapping and phosphoamino acid analysis of phosphorylated VP22 correlated with previously published work. The phosphorylation of virion-associated VP13/14, VP16, and VP22 was demonstrated in cells infected in the presence of cycloheximide. Use of equine herpesvirus 1 in the in vitro release assay resulted in the enhanced release of VP10, the homolog of HSV-1 VP13/14. These results suggest that the dissociation of major tegument proteins from alphaherpesvirus virions in infected cells may be initiated by phosphorylation events mediated by both virion-associated and cellular kinases.

A higher degree of coordination saturation is attainable through the unusual coordination mode in the title compound 1, in which the central pyrazolate groups function both as chelating and as bridging ligands. There is some asymmetry in the bridging, and the N atoms of each μ-η

Beta-catenin is known to associate with the tumour suppressor protein adenomatous polyposis coli (APC), which is highly expressed in developing brain. We have therefore investigated the distribution of beta-catenin and APC in primary cultures of mouse neocortex. Western blotting demonstrated the presence of a single beta-catenin species in our cultures. Immunocytochemistry showed that beta-catenin was plasma membrane associated and concentrated in growth cones in cultured neurons. The APC tumour suppressor protein was also concentrated in growth cones. In glial cells, beta-catenin was localised at cell-cell contacts in a manner similar to that previously described in other cell types. This data suggests a role for both APC and beta-catenin in neuronal growth cones, and for beta-catenin in the formation of cell to cell contacts between glia.

The adenomatous polyposis coli tumour suppressor protein is highly expressed in developing rodent brain, but its function is unclear. Recent studies have suggested a role for this protein in regulating microtubule dynamics. Neuro 2A mouse neuroblastoma cells were previously thought not to express this protein. Using immunochemical techniques, this report corrects this observation. Immunoreactive bands of a size consistent with that of the full-length protein were observed by western blotting. Using immunocytochemistry, punctate immunoreactivity localized to areas of the cell containing microtubules, particularly neurite growth cones, in a distribution suggesting a role in neuritogenesis and growth cone extension. The protein did not localize to actin-rich cellular structures, and perturbation of the actin cytoskeleton had no effect upon this distribution. Treatment of cells with taxol to stabilize microtubules caused the concentration of the immunoreactive puncta to the tips of microtubules and areas along the axis of potential microtubule assembly. Treatment of cells with the microtubule disrupting reagent nocodazole showed that over shorter times the punctate distribution was not dependent upon polymerized microtubules. However, at longer incubation times a decrease in punctate immunostaining was observed. These results indicate that the intracellular distribution of the adenomatous polyposis coli protein is dependent upon microtubule but not actin dynamics. A role for this protein in the regulation of directed microtubule assembly is suggested.

The herpes simplex virus type 1 (HSV-1) capsid protein VP24 (encoded by UL26) was expressed as a GST-fusion protein and used to prepare a group of monoclonal antibodies. These were used to characterize the protein in capsids and virus infected cells and demonstrated that it exists as two polypeptide species. The nature of the relationship between these two species was investigated and found to be associated with disulphide bonding. Under non-reducing conditions a species corresponding to dimers of VP24 was identified in preparations of B capsids, the site of action of the proteinase. Biochemical subcellular fractionation studies suggested that only cleaved forms of UL26 and UL26.5 gene products could be detected in the nucleus of the infected cell at early times post-infection.

Background: Although still largely unexplored, long non-coding RNAs (lncRNAS) are emerging as an integral functional component of the human transcriptome. LncRNAs are mRNA-like transcripts of at least 200 nucleotides (nts) with no protein-coding capacity. Similar to their protein-coding counterparts, lncRNAs are frequently spliced and polyadenylated, but act at the RNA level. The range of functions described for lncRNAs is extensive, and includes key biological roles in chromatin remodeling, alternative splicing and mRNA degradation. Given their biological functions, dysregulation of lncRNAs is rising as an important feature of many disorders, including malignant transformation. However, the extent of the contribution of differential lncRNA expression to normal lung tissue and lung cancer has not been investigated in a comprehensive manner. Hypothesis: We hypothesized that lncRNAs are expressed in a lung tissue-specific manner and that non-small-cell lung cancer (NSCLC) exhibits aberrant lncRNA expression patterns. Methods: Serial Analysis of Gene Expression (SAGE) libraries were used to characterize polyadenylated transcripts in lung tissue compared to a panel of 25 different normal human tissues, and to a cohort of 12 NSCLCs. To generate lncRNA expression profiles, we developed a lncRNA discovery pipeline to map-tag-to-lncRNA matches. To identify differentially expressed lncRNAs we used a permutation test based statistical analysis. Expression pattern in lung tumors were compared to profiles from a variety of cancer types in order to identify lncRNA changes prominent in lung cancer. Results: Here we show that large-scale expression profiling through SAGE, is an effective resource for investigating the expression pattern of polyadenylated lncRNAs. Applying a novel lncRNA discovery pipeline we reveal extensive, tissue-specific lncRNA expression in normal lung compared to a panel of several different normal human tissues. Importantly, our study reveals that NSCLC demonstrate significantly altered lncRNA expression patterns and identify highly dysregulated transcripts associated with this malignancy as oppose to other types of cancer. Conclusion: Collectively, our findings support an important role for tissue-specific lncRNAs in lung cancer. Characterization of the functional role of these transcripts will have a considerable impact on our understanding of lung cancer development and progression, and may reveal clinically important biomarkers.

The compound (ButO)3WN serves to exchange the nitrogen atoms between nitriles (MeCN and PhCN), itself and (ButO)3MoN in solution at room temperature.

Ar3SnX, where Ar = p-MeC6H4, p-CF3C6H4 or Ph and X = OBut or NMe2, are catalyst precursors for the ring-opening of lactides in benzene at 80 °C and the rate of ring-opening of lactides and a variety of related cyclic monomers is influenced by Ar and X such the chemistry of the ring-opening event and the initially formed product may be examined.

A linear supramoleculecular array of anions and cations is present in the solvent-free potassium salt of the homoleptic erbium pyrazolate complex [K{Er(η2 -tBu2 pz)4 }n ] (1). Treatment of 1 with [18]crown-6 in toluene/dimethoxyethane leads to formation of a charge-separated salt [Eq. (a)] in which the methyl group of a toluene molecule interacts with the potassium ion. tBu2 pz=3,5-di-tert-butylpyrazolate.

A higher degree of coordination saturation is attainable through the unusual coordination mode in the title compound 1, in which the central pyrazolate groups function both as chelating and as bridging ligands. There is some asymmetry in the bridging, and the N atoms of each μ-η2 :η2 -pyrazolato ligand are 0.07-0.11 A closer to one of the two Yb centers.

Der Koordinationsabsattigung dient die ungewohnliche Bindungsweise in der Titelverbindung 1, in der die zentralen Pyrazolatgruppen sowohl als chelatisierende wie auch als verbruckende Liganden fungieren. Die Verbruckung weist eine leichte Asymmetrie auf, da die N-Atome der beiden μ-(η2:η2)-Pyrazolatliganden um jeweils 0.07–0.11 A naher an einem der beiden Yb-Zentren sind.

Nat. Genet. 37, 353–355 (2005). The name of the fourth author was incorrect. The correct name is Sofia B. Lizarraga.

The preparation and characterization (IR, 1H, 13C{1H}, 119Sn NMR spectroscopy, elemental analysis and single crystal X-ray structure determination) are reported for Ph3SnOCMe2C(O)OEt (1) and Ph2Sn[OCMe2C(O)NMe2]2 (2). In the solid state, compound 1 contains four-coordinate tin with evidence for incipient bond formation to the ester oxygen: Sn⋯O = 2.648(2) A. Compound 2 contains six-coordinate tin in a pseudo-octahedral geometry. The OCMe2C(O)NMe2 groups form cis-chelates with short, ca. 2.03 A, and long, ca. 2.26 A, Sn–O bonds to alkoxide and amide oxygen atoms, respectively. In solution, compound 1 remains four-coordinate but compound 2 exists as an equilibrium mixture of six-coordinate and five-coordinate species as judged by NMR spectroscopy. At −50 °C in toluene-d8, the six-coordinate isomer is favored and the NMR data are consistent with the structure observed in the solid state. At +50 °C, the NMR data are consistent with a five-coordinate species in which reversible chelation of η2- and η1-OCMe2C(O)NMe2 is fast on the NMR time scale. The molecular structure of 2 and its dynamic solution behavior is proposed to resemble that of Ph2Sn[OCHMeC(O)NMe2]2 formed in the polymerization of L-lactide by Ph2Sn(NMe2)2. The high formation tendency of this compound is proposed to be responsible for the preferential formation of cyclic lactide oligomers (LA/2)n by intrachain transesterification, in contrast to polymerizations employing Ph2Sn(OPri)2, which produce long chains of H–(LA/2)n–OPri where LA = [OCHMeC(O)OCHMeC(O)]. The kinetics of the reactions between Ph3SnX and each of Me2CHC(O)OMe, Me(MeO)CHC(O)OEt and Ph3SnOCHMeC(O)OEt, have been determined from NMR studies in benzene-d6 where X = NMe2 or OPri. Similarly, the reaction between Ph3SnOBut and (p-tolyl)3SnOPri has been followed. The former reactions represent transamidation and transesterification, and the latter models chain transfer. These findings, when compared to the earlier studies of the ring-opening of lactide and its subsequent ring-opening polymerization, indicate that the rate follows the order: chain transfer > ring-opening > ring-opening polymerization > transesterification, although the latter is influenced by the ester end-group.